Genotyping by mass spectrometric analysis of allelic fragments

ABSTRACT

The present invention relates to a method for genotyping a diploid organism by cleaving segments of two alleles such that 7-20 nucleotide fragments that contain a suspected polymorphic locus are produced and comparing the masses of those fragments.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/394,467to Stanton, Wolfe, and Verdine, filed Sep. 10, 1999 now U.S. Pat. No.6,566,059, entitled “A METHOD FOR ANALYZING POLYNUCLEOTIDES.” Ser. No.09/394,467 in turn claims the benefit of U.S. Provisional PatentApplication, serial No. 60/102,724, filed Oct. 1, 1998, and U.S.Provisional Patent Application, serial No. 60/149,533, filed Aug. 17,1999, both of which are also entitled “A METHOD FOR ANALYZINGPOLYNUCLEOTIDES.” Each of these applications is incorporated byreference in its entirety, including drawings and tables, as if fullyset forth herein.

FIELD OF THE INVENTION

The present invention relates generally to organic chemistry, analyticalchemistry, biochemistry, molecular biology, genetics, diagnostics andmedicine. In particular, it relates to methods for genotyping diploidcells or organisms by comparing the masses of fragments of alleles thatinclude the polymorphic locus.

BACKGROUND OF THE INVENTION

The following is offered as background information only and is notintended nor admitted to be prior art to the present invention.

The ability to detect DNA sequence variances in an organism's genome hasbecome an important tool in the diagnosis of diseases and disorders andin the prediction of response to therapeutic regimes. It is becomingincreasingly possible, using early variance detection, to diagnose andtreat, even prevent the occurrence of, a disease or disorder before ithas physically manifested itself. Furthermore, variance detection can bea valuable research tool in that it may lead to the discovery of geneticbases for disorders the cause of which were hitherto unknown or thoughtto be other than genetic.

The most common type of sequence variance is the single nucleotidepolymorphism or SNP. As the name suggests, a SNP involves thesubstitution of one nucleotide to another at a particular locus in agene. While each SNP involves but one nucleotide, a single gene maycontain numerous SNPs.

It is estimated that SNPs occur in human DNA at a frequency of about 1in 100 nucleotides when 50 to 100 individuals are compared. Nickerson,D. A., Nature Genetics, 1998, 223-240. This translates to as many as 30million SNPs in the human genome. However, very few SNPs have any effecton the physical well-being of humans. Detecting the 30 million SNPs andthen determining which of them are relevant to human health will clearlybe a formidable task.

Complete DNA sequencing is presently the definitive procedure fordetecting sequence differences. However, current DNA sequencingtechnology is costly, time consuming and, in order to assure accuracy,highly redundant. Most sequencing projects require a 5- to 10-foldcoverage of each nucleotide to achieve an acceptable error rate of 1 in2,000 to 1 in 10,000 bases. In addition, DNA sequencing is aninefficient way to detect SNPs. While on the average a SNP occurs oncein about every 100 nucleotides, when variance between two copies of agene, for example those associated with two chromosomes, is beingcompared, a SNP may occur as infrequently as once in 1,000 or morebases. Thus, only a small segment of the gene in the vicinity of the SNPlocus is really of interest. If full sequencing is employed, atremendous number of nucleotides will have to be sequenced before anyuseful information is obtained. For example, to compare ten versions ofa 3,000 nucleotide DNA sequence for the purpose of detecting fourvariances among them, even if only 2-fold redundancy is employed (eachstrand of the double-stranded 3,000 nucleotide DNA segment from eachindividual is sequenced once), 60,000 nucleotides would have to besequenced (10×3,000×2). Furthermore, sequencing problems are oftenencountered that can require even more runs, often with new primers.Thus, as many as 100,000 nucleotides might have to be sequenced todetect four variances.

Determination of whether a particular gene of a species or of anindividual of that species contains a SNP is called genotyping. Completesequencing is, therefore, a method for accomplishing genotyping but, asis indicated above, it is slow, costly and extremely inefficient.

An alternative to complete sequencing to compare the masses of fragmentsof two alleles of a gene known or suspected to contain a SNP. A massdifference between any of the fragments indicates that the allelescontained different nucleotides in the divergent fragments which, inturn, reveals that the alleles are heterozygous. Generally, theprocedure involves amplifying a segment of each allele using a modifiednucleotide corresponding to one of the natural nucleotides involved inthe polymorphism, which modified nucleotide imparts enhancedsusceptibility to cleavage at its sites of incorporation, The modifiednucleotide is incorporated into the amplicon in at least a portion ofthe points of occurrence of the natural nucleotide. The modifiedsegments are then cleaved at the sites of incorporation of the modifiednucleotide to give two sets of fragments, which are compared asindicated above. While providing a vast improvement in terms of speed,efficiency and cost over complete sequencing, this procedure is not freeof potential shortcomings.

For example, large differences in assay signals between thermocyclerscan limit the robustness of the procedure. A relatively high occurrence(as much as 25%) of allele-specific reactions in which only onediagnostic product is produced from a heterozygous mixture may confoundthe result. An amplification bias for the allele that has the site ofincorporation of the modified nucleotide farthest from the extendingprimer terminus (called skewing) may occur. In addition, automatedcalling of genotype may be affected by the exponential decrease in massspectometric signal with linear increases in fragment size.Heterozygotes that give fragments differing in size by 5-10 nucleotidescan produce peaks of very unequal intensity that are difficult forautomated devices to recognize.

What is needed, then, is a method that retains the rapid, inexpensive,efficient, yet accurate characteristics of the mass comparison techniquebut which eliminates the above potential shortcomings. The presentinvention provides such a method.

SUMMARY OF THE INVENTION

Thus in one aspect the present invention relates to a method forgenotyping a diploid organism. The method comprises taking two allelesof a target gene of a diploid organism suspected to contain apolymorphism and obtaining a segment of each that contains the suspectedpolymorphic locus. A natural nucleotide is replaced at greater than 90%of its points of occurrence in the two segments with a modifiednucleotide to give two modified segments. In doing so, the naturalnucleotide that is replaced is not a nucleotide involved in thepolymorphism. Furthermore, replacing the natural nucleotide with amodified nucleotide comprises amplification using a primer thathybridizes to each segment such that, after amplification, a firstmodified nucleotide is incorporated between the end of the primer andthe polymorphic locus and a second modified nucleotide is located from 5to 20 nucleotides downstream from the first modified nucleotide. Themodified segments are then cleaved at greater than 90% of the points ofoccurrence of the modified nucleotide to give two sets of fragments eachof which includes a 5-20 nucleotide fragment. The masses of the 5-20nucleotide fragments from the two modified segments are then compared todetect the presence or absence of the polymorphism.

In an aspect of this invention, the second modified nucleotide is from 7to 20 nucleotides downstream of the first modified nucleotide and it isthese 7-20 20 nucleotide fragments that are compared to detect thepresence or absence of the polymorphism.

In as aspect of this invention, the second modified nucleotide is from 7to 12 nucleotides downstream of the first modified nucleotide and it isthese 7-12 nucleotide fragments that are compared to detect the presenceor absence of the polymorphism.

In an aspect of this invention, if there would be less than 5nucleotides between the first and second modified nucleotides, themethod further comprises using a primer that contains a point mutationthat removes the site of incorporation of one of the modifiednucleotides.

In an aspect of this invention, if there are less than 7 nucleotidesbetween the first and the second modified nucleotides, the methodfurther comprises using a primer that contains a point mutation thatremoves the site of incorporation of one of the modified nucleotides.

In an aspect of this invention, if there would be more than 20nucleotides between the first and second modified nucleotides, themethod further comprises a primer, which contains a point mutation thatincorporates a modified nucleotide downstream of the first modifiednucleotide or upstream of the second modified nucleotide.

In an aspect of this invention, if there would be more than 12nucleotides between the first and the second modified nucleotides, themethod further comprises a primer, which contains a point mutation thatincorporates a modified nucleotide downstream of the first modifiednucleotide or upstream of the second modified nucleotide.

An aspect of this invention is a method for genotyping a diploidorganism, comprising, first, providing two alleles of a target gene of adiploid organism suspected to contain a polymorphism and then obtaininga segment from each allele wherein the segment contains the suspectedpolymorphic locus. A natural nucleotide in the segment is then replacedat greater than 90% of its points of occurrence in the two segments witha modified nucleotide to give a first and a second modified segment. INthis aspect of the invention, the natural nucleotide that is replaced isa nucleotide involved in the polymorphism. Replacing the naturalnucleotide with a modified nucleotide comprises amplification using aprimer that hybridizes to each segment such that, after amplification,the suspected polymorphic locus is the first site of incorporation of amodified nucleotide after the end of the primer. Furthermore a secondmodified nucleotide must be located from 5 to 20 nucleotides downstreamof the first modified nucleotide. The first and second modified segmentsare cleaved at greater than 90% of the points of occurrence of themodified nucleotide to give a first and second set of fragments.Finally, the masses of the two sets of fragments are compared for thepresence of the 5-20 nucleotide fragment wherein, if the fragment ispresent or absent in both sets, the gene is homozygous and if thefragment is present in only one set, the gene is heterozygous.

In an aspect of this invention, a nucleotide known to be involved in thepolymorphism is replaced with a mass-modified nucleotide.

In an aspect of this invention, comparing the masses of the fragmentscomprises using a mass spectrometer.

In an aspect of this invention, the mass spectrometer is a MALDI massspectrometer.

In an aspect of this invention, the MALDI mass spectrometer is aMALDI-TOF mass spectrometer.

In an aspect of this invention, the mass spectrometer is an ESI massspectrometer.

In an aspect of this invention, the percentage replacement of a naturalnucleotide with a modified nucleotide, the percentage cleavage at amodified nucleotide, or both the percentage replacement and thepercentage cleavage, is greater than 95%.

In as aspect of this invention, the percentage replacement of a naturalnucleotide with a modified nucleotide, the percentage cleavage at amodified nucleotide, or both the percentage replacement and thepercentage cleavage, is greater than 99%.

DETAILED DESCRIPTION OF THE INVENTION BRIEF DESCRIPTION OF THE FIGURES

The figures herein are provided for the sole purpose of illustratingcertain embodiments of this invention. They are not intended, nor arethey to be construed, as limiting the scope of this invention in anymanner whatsoever.

FIG. 1 illustrates the method of this invention when, after replacementof a nucleotide not involved in the target SNP with a modifiednucleotide followed by cleavage, a fragment of the presently preferredlength is obtained.

FIG. 2 illustrates the method of this invention wherein neither non-SNPnucleotide will result in a fragment that is 12 or fewer nucleotides inlength and a primer is used to artificially create such a fragment.

FIG. 3 illustrates the method of this invention wherein neither non-SNPnucleotide will result in a fragment that is 7 nucleotides or more inlength and a primer is used to artificially create such a fragment.

FIG. 4 illustrates the method of this invention wherein one of the SNPnucleotides is 7 or more and 12 or less nucleotides from the nextoccurrence of the same nucleotide, so that SNP nucleotide is replacedwith a modified nucleotide. After cleavage, rather than obtaining twofragments of equal length but different mass, if the SNP nucleotide ispresent in one of the alleles, a fragment of the desired length will beobtained while, if it is not, the fragment in the vicinity of the SNPwill be substantially longer in that it will include the primer.

DEFINITIONS

As used herein, a “gene” refers to the basic unit of heredity thatcarries the code for every physical trait that distinguishes anindividual of a species.

As used herein, “genotyping” refers to the process of determining thesingle nucleotide polymorphisms (SNPs) present in the genes of a speciesor of an individual of that species.

As used herein, “diploid” refers to an organism in which each gene hastwo alleles, one on each chromosome of a homologous pair.

As used herein, an “organism” refers to any living entity comprised ofeukaryotic cells. This includes plants, reptiles, fish, birds, animalsand, in particular, human beings.

As used herein, an “allele” refers to an alternative form of a gene.More specifically, an allele is one of two or more different versions ofa gene that can occupy the same position or locus on a chromosome. Ifthe same allele occupies the position on both chromosomes of a diploidpair, that DNA, cell or individual is said to be “homozygous.” In, onthe other hand, the alleles occupying the same locus on the twochromosomes are different, the DNA, cell or individual is said to be“heterozygous.”

As used herein, a “reagent” refers to a chemical entity or physicalforce that cleaves a modified segment at the site of incorporation of amodified nucleotide. Such reagents include, without limitation, achemical or combination of chemicals, normal or coherent (laser) visibleor uv light, heat, high energy ion bombardment and irradiation. A“reagent” can refer to a single chemical entity or physical force, acombination of two or more chemical entities or physical forces or acombination of chemical entities and physical forces. If more than onechemical entity or physical force is used, they can be appliedsimultaneously or sequentially. By simultaneously is meant that two ormore reagents are placed in the reaction mixture at the same time with asegment to be cleaved. It is understood that, once placed together inthe reaction mixture, one of the reagents may in fact react with thesegment before the other one. By sequentially is meant that a segment tobe cleaved are first placed in contact with one reagent and only afterthat reagent has performed is the reaction product of the first reactionplaced in contact with the second reagent, etc.

As used herein, a “single nucleotide polymorphism” or “SNP” refers topolynucleotide that differs from another polynucleotide at a particularlocus by virtue of a single nucleotide exchange. A polynucleotide may,of course, contain numerous SNPs; however, each must occur at adifferent locus. For example, exchanging one A for one C, G or T at aparticular locus in the sequence of a polynucleotide constitutes a SNP.When referring to SNPs, the polynucleotide is most often genetic DNA.SNPs can occur in coding and non-coding regions of the gene. Those incoding regions are of primary interest because it is they that can causechanges in the phenotype, i.e., an detectable physical difference in anindividual compared to the general population. Detectable physicaldifferences include, without limitation, a difference in susceptibilityto a particular disease or disorder or a difference in response to atherapeutic regime used to treat or prevent a disease or disorder.

As used herein, a “polymorphic locus” refers to a location in thenucleotide sequence of the alleles of a gene of a diploid organism thatmay be occupied by different nucleotides. The difference may be theresult of a SNP, a point mutation, a nucleotide insertion or anucleotide deletion.

As used herein, a “suspected” polymorphic locus refers to a site in thealleles of a gene where a polymorphism is generally known to occur butit is unknown if the alleles of that gene in the single specificorganism being examined contain the polymorphism.

As used herein, “amplifying” or “amplification” refers to the process ofproducing multiple copies of a segment of a double strandedpolynucleotide by hybridizing natural nucleotide primers 5′ to thesegment to be amplified and then using a polymerase or polymerases toextend the primer to reproduce the sequences of the strands. A commonamplification technique is PCR, the well-known polymerase chainreaction, which results in a logarithmic increase in the number ofcopies of the segment being amplified. The end result of amplificationis the production of a sufficient amount of the segments to permitrelatively facile manipulation. Manipulation refers to both physical andchemical manipulation, that is, the ability to move bulk quantities ofthe segments around and to conduct chemical reactions with them thatresult in detectable products.

As used herein, “downstream” means in the direction away from the 3′ endof a primer, that is, in the direction of extension and “upsteam” meansin a direction toward the 3′ end of a primer or counter to the directionof extension.

As used herein, a “point mutation” refers to a change at a single locusin a polynucleotide strand. The change may be the deletion of anucleotide, the addition of a nucleotide or the substitution of onenucleotide for another.

As used herein a “segment” of an allele refers to a portion of thecomplete nucleotide sequence of the allele.

As used herein a “modified segment” refers to a segment in which anatural nucleotide has been replaced at greater than 90%, preferablygreater than 95%, most preferably greater than 99% of its points ofoccurrence in the segment with a modified nucleotide. For the purposesof this disclosure, the phrase “at substantially each point ofoccurrence” will be used as a short-hand for the preceding list ofincreasing preferences.

As used herein, to “contain” a suspected polymorphic locus means thatthe SNP site is contained in the nucleotide sequence of the amplifiedsegments.

By “homozygous” is meant that the two alleles of a diploid cell ororganism have exactly the same nucleotide sequence.

By “heterozygous” is meant that the two alleles of a diploid cell ororganism have a difference in their nucleotide sequence at a particularlocus. In most cases, the difference is a SNP, although it may be amutation, an insertion or a deletion.

A “sequence” or “nucleotide sequence” refers to the order of nucleotideresidues in a nucleic acid.

A “nucleoside” refers to a base covalently bonded to a sugar moiety. Thebase may be adenine (A), guanine (G) (or its substitute, inosine (I)),cytosine (C), or thymine (T) (or its substitute, uracil (U)). The sugarmay be ribose (the sugar of a natural nucleotide in RNA) or2-deoxyribose (the sugar of a natural nucleotide in DNA).

A “nucleoside triphosphate” refers to a nucleoside linked to atriphosphate group (O⁻—P(═O)(O⁻)—O—P(═O)(O⁻)—O—P(═O)(O⁻)—O—nucleoside).The triphosphate group has four formal negative charges that requirecounter-ions, i.e., positively charged ions. Any positively charged ioncan be used, e.g., without limitation, Na⁺, K⁺, NH₄ ⁺, Mg²⁺, Ca²⁺, etc.Mg²⁺ is one of the most commonly used counter-ions. It is acceptedconvention in the art to omit the counter-ion, which is understood to bepresent, when displaying nucleoside triphosphates; the convention isfollowed in this application.

As used herein, unless expressly noted otherwise, the term “nucleosidetriphosphate” or reference to any specific nucleoside triphosphate;e.g., adenosine triphosphate, guanosine triphosphate or cytidinetriphosphate, refers to the triphosphate made using either aribonucleoside or a 2′-deoxyribonucleoside.

A “nucleotide” refers to a nucleoside linked to a single phosphategroup.

A “natural nucleotide” refers to an A, C, G or U nucleotide whenreferring to RNA and to dA, dC, dG and dT (the “d” referring to the factthat the sugar is deoxyribose) when referring to DNA. A naturalnucleotide also refers to a nucleotide which may have a differentstructure from the above, but which is naturally incorporated into apolynucleotide sequence by the organism which is the source of thepolynucleotide.

As used herein, a “modified nucleotide” refers to a nucleotide thatmeets two criteria. First, a modified nucleotide is a “non-natural”nucleotide. In one aspect, a “non-natural” nucleotide may be a naturalnucleotide that is placed in non-natural surroundings. For example, in apolynucleotide that is naturally composed of deoxyribonucleotides, i.e.,DNA, a ribonucleotide would constitute a “non-natural” nucleotide.Similarly, in a polynucleotide that is naturally composed ofribonucleotides, i.e., RNA, a deoxyribonucleotide would constitute anon-natural nucleotide. A “non-natural” nucleotide also refers to anatural nucleotide that has been chemically altered. For example,without limitation, one or more substituent groups may be added to thebase, sugar or phosphate moieties of the nucleotide. Or, one or moresubstituents may be deleted from the base, sugar or phosphate moiety.Finally, one or more atoms or substituents may be substituted for one ormore other atoms or substituents in the nucleotide. A “modified”nucleotide may also be a molecule that resembles a natural nucleotidelittle, if at all, but is nevertheless capable of being incorporated bya polymerase into a polynucleotide in place of a natural nucleotide.

The second criterion associated with a “modified” nucleotide, as theterm is used herein, is that it alters the cleavage properties of thepolynucleotide into which it is incorporated. For example, withoutlimitation, incorporation of a ribonucleotide into a polynucleotidecomposed predominantly of deoxyribonucleotides imparts a heightenedsusceptibility to alkaline cleavage at the site of incorporation thatdoes not otherwise exist. This second criterion of a “modified”nucleotide may be met by a single non-natural nucleotide substitution(e.g., the substitution of a ribonucleotide for a deoxyribonucleotidedescribed above). It may also be met by substitution of two or morenon-natural nucleotides that do not individually alter the cleavageproperties of a polynucleotide but, rather, when in a particular spatialrelationship with one another, result in cleavage of the polynucleotide(referred to as “dinucleotide cleavage”).

As used herein, “having different cleavage characteristics” refers totwo or more modified nucleotides that, when incorporated into apolynucleotide, can be selectively cleaved in each other's presence byusing different reagents and/or reaction conditions.

“Replacing” a natural nucleotide with a modified nucleotide refers tothe process of amplifying a segment using one modified nucleotide andthe three remaining natural nucleotides such that the natural nucleotidecorresponding to the modified nucleotide is replaced at substantiallyeach point of occurrence in the segment by the modified nucleotide. Asused herein, “replacing” may also refer to the process of amplifying asegment using two modified nucleotides and the two remaining naturalnucleotides simultaneously such that each natural nucleotide is replacedas substantially each point of occurrence in the segment by itscorresponding modified nucleotide.

“Cleaving” a modified segment refers to the process of contacting thesegment with a reagent that selectively severs the nucleotide chain atsubstantially each point of occurrence of a modified nucleotide.

A “polynucleotide” refers to a linear chain of 30 or more nucleoside5′-monophosphate residues linked by phosphodiester bonds between the 3′hydroxyl group of one sugar and the 5′ hydroxyl group of the next.

A “modified polynucleotide” refers to a polynucleotide in which anatural nucleotide has been replaced at substantially each point of itsoccurrence with a modified nucleotide. It may also refer to thereplacement of two, three or four natural nucleotides with two, three orfour modified nucleotides where each of the modified nucleotides altersthe cleavage properties of the resulting modified polynucleotidedifferently. Cleavage can then be selectively carried out with eachmodified nucleotide in the presence of the others.

As used herein, to “alter the cleavage properties” of a polynucleotidemeans to render the polynucleotide more or less susceptible to cleavageat the point of incorporation of a modified nucleotide than it would bewith a natural nucleotide or a different non-natural nucleotide at thesame locus. It is presently preferred to “alter the cleavage properties”by rendering a polynucleotide more susceptible to cleavage at the siteof incorporation of a modified nucleotide than at any other locus in themolecule. As used herein, the use of the singular when referring tonucleotide substitution or cleavage is to be construed as includingsubstitution or cleavage at substantially each point of occurrence of amodified nucleotide unless expressly stated otherwise.

As used herein, a “template” refers to a polynucleotide strand, which apolymerase uses as a means of recognizing which nucleotide it shouldnext incorporate into a growing strand to duplicate a polynucleotide. Ifthe polynucleotide is DNA, it may be single-stranded or double-stranded.When employing the polymerase chain reaction (PCR) to amplify a templateusing the methods of this invention, the copies made contain modifiednucleotides. These modified segments are still capable of serving astemplates for the production of further copies of identically modifiedamplicons.

As used herein, a “primer” refers to an oligonucleotide formed fromnatural nucleotides, the sequence of which is complementary to a segmentof a template to be replicated. A polymerase uses the primer as thestarting point for the replication process. By “complementary” is meantthat the nucleotide sequence of a primer is such that it will hybridizeto the template by formation of hydrogen bonded base-pairs over a lengthof at least ten consecutive bases. In the methods of this invention, aprimer is never modified by replacement of a natural nucleotide with amodified nucleotide nor does cleavage ever occur in the nucleotidesequence of the primer.

As used herein, a “polymerase” refers, without limitation, to DNA or RNApolymerases, mutant versions thereof and to reverse transcriptases. DNAor RNA polymerases can be mutagenized by, without limitation, nucleotideaddition, nucleotide deletion, one or more point mutations, “DNAshuffling” or joining portions of different polymerases to make chimericpolymerases. Combinations of these mutagenizing techniques may also beused. A polymerase catalyzes the assembly of nucleotides to formpolynucleotides. Polymerases may be used either to extend a primer onceor repetitively. Repetitive extension is sometimes referred to asamplification. Amplification may be accomplished by, without limitation,PCR, NASBR, SDA (Strand Displacement Amplification), 3SR (Self-SustainedSequence Replication Reaction), TSA (Tyramide Signal Amplification) androlling circle replication. In the methods of this invention, one ormore polymerases and one or more extension or amplification techniquesmay be used to replicate a particular polynucleotide.

As used herein, a “chemical oxidant” refers to a reagent capable ofincreasing the oxidation state of a group on a molecule. For instance,without limitation, a hydroxyl group (—OH) can be oxidized to analdehyde, ketone or acid. Some examples of chemical oxidants are,without limitation, potassium permanganate, t-butyl hypochlorite,m-chloroperbenzoic acid, hydrogen peroxide, sodium hypochlorite, ozone,peracetic acid, potassium persulfate, and sodium hypobromite.

As used herein, a “chemical base” refers to a chemical compound that, inaqueous medium, has a pK greater than 7.0. A chemical base may beinorganic or organic. Examples of inorganic chemical bases include,without limitation, alkali (sodium, potassium, lithium) and alkalineearth (calcium, magnesium, barium) hydroxides, carbonates, bicarbonates,phosphates and the like. Ammonium hydroxide is another inorganicchemical base. Nitrogen-containing organic compounds such as pyridine,aniline, quinoline, morpholine, piperidine and pyrrole are also chemicalbases. Nitrogen-containing chemical bases may be primary (the nitrogenhas two hydrogen atoms and one other substituent on it), secondary (thenitrogen has one hydrogen and two other substituents on it) or tertiary(the nitrogen has no hydrogen atoms on it). Chemical bases may be usedas aqueous solutions, which may be mild (usually due to dilution) orstrong (concentrated solutions). A chemical base also refers to a strongnon-aqueous organic base; examples include, without limitation, sodiummethoxide, sodium ethoxide and potassium t-butoxide.

Secondary amines are presently preferred chemical bases for use in thecleavage of modified nucleotides. Secondary amines useful in the methodsof this invention include, without limitation, pyrrolidine, piperidine,3-pyrrolidinol, 2-pyrrolidinemethanol, 3-pyrrolidinemethanol,4-hydroxypiperidine, 4-(2-hydroxyethyl)piperidine, hexamethyleneimine,heptamethyleneimine, diethylamine, diproylamine, dibutylamine, proline,morpholine, piperizine, picolinic acid, piperazine-2-carboxylic acid,4-piperidineethanol and isopecotic acid. A secondary amine useful in themethods herein may also be polymer bound, for example, withoutlimitation, piperidine-4-carboxylic acid polymine resin (polystyrene).

As used herein, the term “acid” refers to a substance that dissociatesin water to produce one or more hydrogen ions. An acid may be inorganicor organic. It may be a strong acid, which generally infers highlyconcentrated, or mild, which generally infers dilute. It is, of course,understood that acids inherently have different strengths; e.g.,sulfuric acid is much stronger than acetic acid. The proper choice ofacid will be apparent to those skilled in the art from the disclosuresherein. Preferably, the acids used in the methods of this invention aremild. Examples of mild inorganic acids are, without limitation, dilutehydrochloric acid, dilute sulfuric acid, dilute nitric acid, phosphoricacid and boric acid. Examples, without limitation, of mild organic acidsare formic acid, acetic acid, benzoic acid, p-toluenesulfonic acid,trifluoracetic acid, naphthoic acid, uric acid and phenol.

As used herein, “bond,” “bonding” or “bonded” refers, unless otherwiseexpressly stated, to covalent bonds between the entities which are thesubject of the bonding.

As used herein, a “functional group” refers, without limitation, to anentity such as amino (—NH₂), hydroxyl (—OH), cyano (—C≡N), nitro (NO₂),carboxyl (—COOH), acid halide (C(O)X, wherein X is chloro or bromo),ester (—C(O)OR, R is methyl ethyl, etc.), formyl (—CHO), keto(—CH₂C(O)CH₂—), alkenyl (—C═C—), alkynyl (—C≡C—), halo (F, Cl, Br and I)groups and the like, which are capable of reacting with other functionalgroups to form bridges of covalently bonded atoms linking together theentities to which the functional groups were initially bonded. Forinstance, an amino functional group can react with an acid halidefunctional group to form an amide (—C(O)NH₂). Likewise, a hydroxyl groupcan react with an acid halide to form an ester. Many such functionalgroups are known to those skilled in the art. The use of any of them inthe methods herein to link modified nucleotides to fluorescent tags.

“Hybridizing” or “hybridization” refers to the formation of A-T or C-Gbase pairs among a string of contiguous nucleotides in anoligonucleotide or polynucleotide (usually at least 10 to form a stablehybridization product). In the present case, the oligonucleotides are aprimer and a template or a primer and an immobilization oligonucleotide.To hybridize, the primer and template or primer and oligonucleotide mustbe “complementary” in the region of base-pair formation. “Complementary”means that the locus of each A, C, G and T (or U, if the oligonucleotideor template comprises ribonucleotides) in the sequence of thehybridizing portion of the primer corresponds to a T, G, C or A,respectively, in the same locus of the sequence of the template oroligonucleotide.

“Mass spectrometry” refers to a technique for mass analysis known in theart which includes, but is not limited to, matrix assisted laserdesorption ionization (MALDI) and electrospray ionization (ESI) massspectrometry optionally employing, without limitation, time-of-flight,quadrupole or Fourier transform detection techniques. While the use ofmass spectrometry constitutes a preferred embodiment of this invention,it will be apparent that other instrumental techniques are, or maybecome, available for the determination of the mass or the comparison ofmasses of oligonucleotides. An aspect of the present invention is thedetermination and comparison of masses and any such instrumentalprocedure capable of such determination and comparison is deemed to bewithin the scope and spirit of this invention.

As used herein, the terms “selective,” “selectively,” “substantially,”“essentially,” “uniformly” and the like, mean that the indicated eventoccurs to a particular degree. For example, the percent incorporation ofa modified nucleotide herein is characterized as “substantiallycomplete.” As used herein, this means greater than 90%, preferablygreater than 95% and, most preferably, greater than 99%. With regard tocleavage at a modified nucleotide, “selectively” means greater than 10times, preferably greater than 25 times, most preferably greater than100 times that of the natural nucleotide in the modified polynucleotide.The percent cleavage at a modified nucleotide is also referred to hereinas being “substantially complete.” This means greater than 90%,preferably greater than 95%, most preferably greater than 99% complete.

Discussion

The methods of this invention can be used to examine the genetic DNA ofan individual displaying symptoms of a particular disease or disorderknown or suspected to be genetically based. Comparison of the DNA of theindividual with that of healthy members of the same population willconfirm whether the individual is afflicted with a particulargenetically-related disease or disorder. Conversely, the method can beused to study an individual displaying symptoms of a disease or disorderof unknown origin to determine if it has a genetic component.

Of course, the methods herein are not limited to the examination of thegenetic aspects of diseases and disorders of human beings. For example,without limitation, plants have genetic variations that affect suchtraits as disease resistance, temperature accommodation, droughtresistance, product size, crop yield, flavor, etc. Animals likewise havegenetic variations that affect size, fertility, growth rate, diseaseresistance, body composition and the like. Knowing which geneticvariations are responsible for these and may other beneficialcharacteristics can have significant economic impact. The methods ofthis invention are equally applicable to these areas of genetic inquiry.

Nucleotide Modification and Cleavage

A modified nucleotide may contain a modified base, a modified sugar, amodified phosphate ester linkage or any combination of these. Withregard to the present invention, the presently preferred modifiednucleotide is a base-modified nucleotide.

Base-modified Nucleotides

Base-modified nucleotide refers to the chemical modification of theadenine, cytosine, guanine or thymine (or, in the case of RNA, uracil)moiety of a nucleotide. The resulting modified nucleotide is moresusceptible to cleavage than the natural nucleotides in thepolynucleotide. The following are examples, without limitation, of basemodification. Other modifications of bases will become apparent to thoseskilled in the art based on the disclosures herein. Such basemodifications are within the scope of this invention.

1. Adenine (1) can be replaced with 7-deaza-7-nitroadenine (2).7-Deaza-7-nitroadenine is readily incorporated into polynucleotides byvarious polymerases. The 7-nitro group activates C-8 to attack bychemical base such as, without limitation, aqueous sodium hydroxide oraqueous piperidine, which results in strand scission. Verdine, et al.,JACS, 1996, 118:6116-6120;

When the cleavage reaction is carried out in the presence of aphosphine, for example, without limitation, tris(2-carboxyethyl)phosphine (TCEP) and a base, complete cleavage is obtained. Thus, whenDNA modified by incorporation of 7-nitro-7-deaza-2′-deoxyadenosine wastreated with 0.2 M TCEP/1 M piperidine/0.5 M Tris base at 95° C. for onehour, complete cleavage was observed on denaturing polyacrylamide gel(20%) electrophoresis. Other bases such as, without limitation, ammoniumhydroxide can be used in place of piperidine and Tris base. Thisprocedure, i.e., the use of a phosphine in conjunction with a base,should work for any base-modified nucleotide in which the modifiedadenine, cytosine, guanine, thymine or uracil is labile to chemicalbase.

Secondary amines are presently preferred chemical bases for use incleavage reactions of this invention. Some representative secondaryamines useful in cleavage reactions of this invention include, withoutlimitation, diethylamine, dipropylamine and pyrrolidine. However,secondary amines having a boiling point above 100° C. at atmosphericpressure are preferred. While not being bound to any particular theory,this might be due to the fact that lower boiling secondary amines arevolatilized at the temperatures used for cleavage, 90° C. or higher,making it difficult to maintain an optimal concentration of the amine inthe cleavage reaction. Examples of higher boiling secondary aminesinclude, without limitation, dibutylamine, piperidine, 3-pyrrolidinol,hexamethyleneimine, morpholine and pyrazine. Secondary amines having aboiling point above 150° C. are even more preferable, with those havinga boiling point above 200° C. being the presently most preferred. Suchhigh boiling secondary amines include, without limitation,heptamethyleneimine, 3-pyrrolidinol, 2-pyrrolidinemethanol,3-pyrrolidinemethanol, proline, picolinic acid, piperazine-2-carboxylicacid, 4-piperidineethanol, isonipecotic acid and piperidine-4-carboxlicacid polymine resin (polystyrene). 3-Pyrrolidinol,2-pyrrolidinemethanol, 3-pyrrolidinemethanol and piperidine-4-ethanol(4-(2-hydroxyethyl)piperidine) are presently preferred high boilingsecondary amines for use in the methods of this invention.

When the cleavage reaction is carried out in the presence of a phosphineand a base, a unique adduct forms. For example, when the phosphine is,without limitation, tris(2-carboxyethyl) phosphine (TCEP), massspectrometry of the product is consistent with a structure having aribose-TCEP adduct at its 3′ end and a phosphate moiety at its 5′ end:

The mechanism of formation of the phosphine adduct is not presentlyknown; however, without being held to any particular theory, thefollowing is a possibility:

The incorporation of a phosphine into the cleavage product can be usedto label polynucleotide fragments at the same time cleavage is beingperformed. By using a phosphine that contains a label or tag but isstill capable of forming the above-described adduct, such entities as,without limitation, mass tags, fluorescence tags, radioactive tags andion-trap tags can be incorporated directly into polynucleotide fragmentsduring cleavage.

While other phosphines useful in the cleavage procedure described abovewill become apparent to those skilled in the art based on thedisclosures herein, and therefore are within the scope of thisinvention, TCEP is presently preferred. The carboxy (—C(O)OH) groups ofTCEP can be readily modified, for example, without limitation, byreaction with an amine, alcohol or mercaptan to form an amide, ester ormercaptoester:

In the above scheme, M¹ and M² are independently oxygen, —NH, NR¹ orsulfur. R¹ and R² are independently mass, fluorescent, radioactive orion trap tags.

When a carboxy group is reacted with a carbodiimide in the absence of anucleophile, the product rearranges to form an N-acylurea. If thecarbodiimide contains a fluorophore, the phosphine will then carry it:

Amino group-containing fluorophores such as fluoresceinyl glycine amide,(5-aminoacetamido)fluorescein, 7-amino-4-methylcoumarin,2-aminoacridone, 5-aminofluorescein, 1-pyrenemethylamine and5-aminoeosin may also be used to prepare labeled phosphines. Aminoderivatives of Lucifer Yellow and Cascade Blue can also be employed ascan amino derivatives of biotin. In addition, hydrazine derivatives suchas rhodamine and Texas Red hydrazine may be useful in this method.

Fluorescent diazoalkanes, such as, without limitation,1-pyrenyldiazomethane, may be used to form esters with TCEP. Fluorescentalkyl halides may also react with the carboxylate anion (—C(O)O⁻) of thephosphine to form esters. Such halides include, without limitation,panacyl bromide, 3-bromoacetyl-7-diethylaminocoumarin,6-bromoacetyl-2-diethylaminonaphthalene, 5-bromomethylfluorescein,BODIPY® 493/503 methyl bromide, monobromobimanes and iodoacetamides suchas coumarin iodoacetamide. Naphthalimide sulfonate ester reacts rapidlywith the anions of carboxylic acids in acetonitrile to give adductswhich are detectable by absorption at 259 nm down to 100 femtomoles andby fluorescence at 394 nm down to four femtomoles.

There are also many amine-reactive fluorescent probes known in the art.TCEP is readily converted into a primary amine and reacted with theseentities:

Cytosine (4) can be replaced with 5-azacytosine (5). 5-Azacytosine canbe incorporated into polynucleotides by polymerases. 5-Azacytosine issusceptible to cleavage by chemical base, particularly aqueous base suchas aqueous piperidine or aqueous sodium hydroxide. Verdine, et al.,Biochemistry, 1992, 31:11265-1123

3. Guanine (6)can be replaced with 7-methylguanine (7) and can likewisebe readily incorporated into polynucleotides by polymerases (Verdine, etal., JACS, 1991, 113:5104-5106). The resulting nucleotide is susceptibleto attack by chemical base, such as, without limitation, aqueouspiperidine (Siebenlist, et al., Proc. Natl. Acad. Sci. USA, 1980,77:122).

4. Either thymine (9) or uracil (10) may be replaced with5-hydroxyuracil (11) (Verdine, JACS, 1991, 113:5104) or 5-aminouracil(12). As with the above-modified bases, these nucleotides can beincorporated into a polynucleotide by enzyme-catalyzed polymerization.While not absolutely necessary, in a presently preferred embodiment,cleavage of 5-hydroxyuracil is accomplished by first treating it with anoxidizing agent, for instance, aqueous permanganate, and then with achemical base such as aqueous piperidine, as shown.

5. Pyrimidines substituted at the 5-position with an electronwithdrawing group such as, without limitation, nitro, halo or cyano,should be susceptible to nucleophilic attack at the 6-position followedby base-catalyzed ring opening and subsequent degradation of thephosphate linkage. An example, which is not to be construed as limitingthe scope of this invention in any manner, using 5-substituted cytidineis shown below. If the cleavage is carried out in the presence oftris(carboxyethyl)phosphine (TCEP), adduct 10 may be obtained. The TCEPmay be functionalized with a fluorophore as discussed above.

Genotyping

As DNA sequence data accumulates for various species, particularlyhumans, more and more variances in the genetic code for individualscompared to the general population within a species are beingrecognized. Some of these variances are being related to phenotypicdifferences such as an increased susceptibility to a particular diseaseor a different reaction to a given therapeutic regime. Thus, there isincreasing demand for automated, accurate, high throughput, inexpensivemethods for determining the status of a specific nucleotide ornucleotides in individuals where variation has been discovered. Thisprocedure—the determination of the nucleotide at a particular locationin a DNA sequence—is referred to as genotyping. The methods of thisinvention are eminently suitable to genotyping.

First, both alleles of a gene that is known to have a polymorphic siteare obtained. Then a segment of each allele that contains thepolymorphic locus is amplified using a modified nucleotide to replace anatural nucleotide at greater than 90% of the points of its occurrencein the segment. Preferably, replacement occurs at greater than 95% and,most preferably, greater than 99% of the points of occurrence of thenatural nucleotide. Amplification by PCR is presently preferred.

In a presently preferred embodiment of this invention, the naturalnucleotide that is replaced is not one that is involved in the suspectedpolymorphism. Thus, if the gene is known to involve an A/T SNP, then themodified nucleotide would be a modified C or G, etc. The modifiednucleotide renders the amplified segments more susceptible to cleavageat its sites of incorporation than elsewhere in the segments.

Which natural nucleotide is replaced with a modified nucleotide isdetermined by examination of the sequence of the segments containing thepolymorphic site. First, a nucleotide is selected such that, when it isreplaced with a modified nucleotide, a modified nucleotide will belocated between the end of the primer and the SNP so that a cleavagesite is generated between the primer and the SNP. This eliminates thepotential problem discussed previously regarding amplification biassince the site of the first modified nucleotide will be the same in eachallele.

In addition, the nucleotide is selected such that, after cleavage of themodified segment, a fragment will be generated that is from 5-20nucleotides long and contains the polymorphic locus. In a presentlypreferred embodiment, the length of the fragment will be from 7-12nucleotides. This is exemplified in FIG. 1.

In FIG. 1, the bicolor diamond represents the SNP, the white upperportion representing one nucleotide involved in the SNP and the hashedlower portion the other. Thus the upper segment represents bothpotential alleles. The numbered circles represent the locations of oneof the nucleotides not involved in the SNP. As indicated, there are 11nucleotides, including the SNP, between nucleotides 1 and 2. When theseare replaced with modified nucleotides and the modified segmentscleaved, two fragments of the same length that may differ only in themass of the nucleotide at the SNP, will be produced. If, on comparison,the masses of these two fragments are the same, the alleles of the genein the particular individual from whom they were obtained arehomozygous. If the masses are different, the alleles are heterozygous.

If it should occur that neither of the nucleotides that are not involvedin the SNP would result in a fragment of from 5 to 20 nucleotides inlength, such a fragment may be created as follows.

If the length of the resulting SNP-containing fragment would be greaterthan 20, then a primer can be used to introduce a nucleotide that willshorten the length of the fragment. That is, a primer can be synthesizedthat hybridizes with the allelic segment such that a modified base isinserted upstream of the SNP in the primer extension product and iswithin 20, preferably within 12, nucleotides of the downstream modifiedbase. This can be accomplished by using a primer that contains a pointmutation, i.e., a nucleotide mismatch, that corresponds to thedownstream modified nucleotide. Cleavage would then occur at theprimer-introduced modified nucleotide and at the downstream modifiednucleotide to give the desired fragment for comparison. This isillustrated in FIG. 2.

In FIG. 2, the bicolor diamond represents the SNP, as above. Thenumbered circles represent the locations of a nucleotide other than oneinvolved in the SNP. As indicated, the number of nucleotides betweennucleotide 1 and nucleotide 2 is greater than 12 nucleotides, thepresently preferred maximum. (For the purpose of this example, it isassumed that the other non-SNP nucleotide would also result in afragment longer than 12 or shorter than 7, nucleotides.) A primer isthus synthesized that will hybridize (indicated by the black arrow, thehead of which points in the direction of primer extension) such that thenucleotide indicated by the black “X” in the primer will become part ofthe primer extension product and will occur within 12 nucleotides ofdownstream modified nucleotide 2. “X” is the same modified nucleotide as2; thus, the requisite distance between modified nucleotides is createdin the primer extension product and the analysis can proceed as above.

If, on the other hand, the SNP-containing fragment would be less than 7,or worse, less than 5, nucleotides in length if either nucleotide thatis not involved in the SNP were replaced with a modified nucleotide, aprimer can be synthesized that eliminates a site of incorporation of amodified nucleotide. This is illustrated in FIG. 3.

In FIG. 3, the bicolor diamond again represents the SNP, the numberedcircles represent one of the nucleotides that is not involved in theSNP. (Again, for the sake of this illustration, it is assumed that theother non-SNP nucleotide would likewise lead to a fragment that wasgreater than or equal to 12 nucleotides long or equal to or shorter than7 nucleotides.) As can be seen, replacement of the uninvolved nucleotidewith a modified nucleotide followed by cleavage would result in afragment that is too small. Thus, the primer synthesized to extend thesegment would be designed to include a base mismatch, indicated by an“X” in the primer sequence, that would replace one of the uninvolvednucleotides with the other uninvolved nucleotide. For example if the SNPis an A/T polymorphism and nucleotides 1 and 2 are C's, then X would bea G and 4 would be a modified C corresponding to nucleotide 3 in thesegment. The primer extension product would therefore contain modifiednucleotides 2 and 4, which are 7 or more and 12 or less nucleotidesapart. Cleavage would then give fragments in the presently preferredrange of this invention.

In the above methods, it may be desirable to increase the potential massdifference between the same-length fragments. This is readilyaccomplished by replacing one of the SNP nucleotides with amass-modified nucleotide, which does not create a cleavage site but onlyalters the molecular weight of that nucleotide and therefore thefragment.

In a further embodiment of this invention, the natural nucleotide thatis replaced is one of the nucleotides involved in the SNP. Otherwise themethod is carried out as indicated above except that the analysis of thefragments involves simply looking for the presence of the 5-20,preferably 7-12, nucleotide fragment. That is, no mass comparison isrequired. This is illustrated in FIG. 4.

In FIG. 4, the bicolor diamond again represents the SNP. The numberedcircles, however, represent the remaining loci of one of the nucleotidesinvolved in the SNP, for the sake of this example, the SNP nucleotiderepresented by the upper white portion of the diamond. As can be seen,the white SNP nucleotide is already 7-12 nucleotides from the next samenucleotide. If the white SNP nucleotide is replaced with its counterpartmodified nucleotide and cleavage performed, a fragment of a presentlypreferred length will be obtained. On the other hand, if the otherallele has the different hashed nucleotide at the SNP locus, the SNPlocus will not be a cleavage site after replacement of the white SNPnucleotide with a modified nucleotide. Thus, after cleavage the hashednucleotide allele will give a much longer fragment that includes theprimer. Thus, all that must be done to genotype the alleles is to lookfor the shorter fragment. That is, if the shorter fragment is present inone of the sets of fragments but not the other, the alleles areheterozygous. If the shorter nucleotide fragment is observed in bothsets of fragments, the alleles are homozygous in the white nucleotide.If the shorter nucleotide fragment is not observed in either set offragments, the alleles must be homozygous in the black nucleotide.

Although certain embodiments and examples have been used to describe thepresent invention, it will be apparent to those skilled in the art thatchanges in the embodiments and examples shown may be made withoutdeparting from the scope of this invention.

Other embodiments are contained within the following claims.

What is claimed:
 1. A method for genotyping a diploid organism,comprising: providing two alleles of a target gene of a diploid organismsuspected to contain a polymorphism; obtaining a first segment of oneallele wherein the segment contains the suspected polymorphic locus;obtaining a second segment from the other allele wherein the segmentalso contains the suspected polymorphic locus; replacing a naturalnucleotide at greater than 90% of its points of occurrence in the firstand the second segment with a modified nucleotide to give a first and asecond modified segment; wherein, the natural nucleotide that isreplaced is not a nucleotide involved in the polymorphism; replacing thenatural nucleotide with a modified nucleotide comprises amplificationusing a primer that hybridizes to each segment such that, afteramplification, a first modified nucleotide is incorporated between the3′ end of the primer and the polymorphic locus; and, a second modifiednucleotide is located from 5 to 20 nucleotides downstream of the firstmodified nucleotide; cleaving the first and second modified segments atgreater than 90% of the points of occurrence of the modified nucleotideto give a first and second set of fragments each of which comprises a5-20 nucleotide fragment; and, comparing masses of the 5-20 nucleotidefragments obtained from the first and the second modified segment todetect the presence or absence of the polymorphism.
 2. The method ofclaim 1, wherein the second modified nucleotide is from 7 to 20nucleotides downstream of the first modified nucleotide; and, the massesof the 7-20 nucleotide fragments obtained from the first and the secondmodified segment are compared to detect the presence or absence of thepolymorphism.
 3. The method of claim 1, wherein the second modifiednucleotide is from 7 to 12 nucleotides downstream of the first modifiednucleotide; and, the masses of the 7-12 nucleotide fragments obtainedfrom the first and the second modified segment are compared to detectthe presence or absence of the polymorphism.
 4. The method of claim 1,wherein, if there would be less than 5 nucleotides between the first andsecond modified nucleotides, the method further comprises using a primerthat contains a point mutation that removes the site of incorporation ofeither one of the modified nucleotides.
 5. The method of claim 1,wherein, if there are would be less than 7 nucleotides between the firstand the second modified nucleotides, the method further comprises usinga primer that contains a point mutation that removes the site ofincorporation of either one of the modified nucleotides.
 6. The methodof claim 1, wherein, if there would be more than 20 nucleotides betweenthe first and second modified nucleotides, the method further comprisesa primer, which contains a point mutation that incorporates a modifiednucleotide downstream of the first modified nucleotide or upstream ofthe second modified nucleotide.
 7. The method of claim 1, wherein, ifthere would be more than 12 nucleotides between the first and the secondmodified nucleotides, the method further comprises a primer, whichcontains a point mutation that incorporates a modified nucleotidedownstream of the first modified nucleotide or upstream of the secondmodified nucleotide.
 8. The method of claim 1, further comprisingreplacing a nucleotide known to occur at the polymorphic site with amass-modified nucleotide.
 9. The method of claim 1, wherein comparingthe masses of the fragments comprises using a mass spectrometer.
 10. Themethod of claim 9, wherein the mass spectrometer is a MALDI massspectrometer.
 11. The method of claim 10, wherein the MALDI massspectrometer is a MALDI-TOF mass spectrometer.
 12. The method of claim9, wherein the mass spectrometer is an ESI mass spectrometer.
 13. Amethod for genotyping a diploid organism, comprising: providing twoalleles of a target gene of a diploid organism suspected to contain apolymorphism; obtaining a first segment of one allele wherein thesegment contains the suspected polymorphic locus; obtaining a secondsegment from the other allele wherein the segment also contains thesuspected polymorphic locus; replacing a natural nucleotide at greaterthan 90% of its points of occurrence in the first and the second segmentwith a modified nucleotide to give a first and a second modifiedsegment; wherein, the natural nucleotide that is replaced is anucleotide involved in the polymorphism; replacing the naturalnucleotide with a modified nucleotide comprises amplification using aprimer that hybridizes to each segment such that, after amplification,the suspected polymorphic locus is the first site of incorporation of amodified nucleotide after the end of the primer; and, a second modifiednucleotide is located from 5 to 20 nucleotides downstream of the firstmodified nucleotide; cleaving the first and second modified segments atgreater than 90% of the points of occurrence of the modified nucleotideto give a first and second set of fragments; and, comparing masses ofthe two sets of fragments for the presence of the 5-20 nucleotidefragment wherein, if the fragment is present or absent in both sets, thegene is homozygous and if the fragment is present in only one set, thegene is heterozygous.
 14. The method of claim 13, wherein the secondmodified nucleotide is from 7 to 20 nucleotides downstream of the firstmodified nucleotide; and, the masses of the two sets of fragmentsobtained from the first and the second modified segment are compared todetect the presence of the 7-20 nucleotide fragment.
 15. The method ofclaim 13, wherein the second modified nucleotide is from 7 to 12nucleotides downstream of the first modified nucleotide; and, the massesof the two sets of fragments obtained from the first and the secondmodified segment are compared to detect the presence of the 7-12nucleotide fragment.
 16. The method of either claim 1 or claim 13,wherein comparing the masses of the fragments comprises using a massspectrometer.
 17. The method of claim 16, wherein the mass spectrometeris a MALDI mass spectrometer.
 18. The method of claim 17, wherein theMALDI mass spectrometer is a MALDI-TOF mass spectrometer.
 19. The methodof claim 16, wherein the mass spectrometer is an ESI mass spectrometer.20. The method of either claim 1 or claim 13, wherein the percentagereplacement of a natural nucleotide with a modified nucleotide, thepercentage cleavage at a modified nucleotide, or both the percentagereplacement and the percentage cleavage, is greater than 95%.
 21. Themethod of either claim 1 or claim 13, wherein the percentage replacementof a natural nucleotide with a modified nucleotide, the percentagecleavage at a modified nucleotide, or both the percentage replacementand the percentage cleavage, is greater than 99%.